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Measuring macromolecular size distributions and interactions at high concentrations by sedimentation velocity.

ABSTRACT: In concentrated macromolecular solutions, weak physical interactions control the solution behavior including particle size distribution, aggregation, liquid-liquid phase separation, or crystallization. This is central to many fields ranging from colloid chemistry to cell biology and pharmaceutical protein engineering. Unfortunately, it is very difficult to determine macromolecular assembly states and polydispersity at high concentrations in solution, since all motion is coupled through long-range hydrodynamic, electrostatic, steric, and other interactions, and scattering techniques report on the solution structure when average interparticle distances are comparable to macromolecular dimensions. Here we present a sedimentation velocity technique that, for the first time, can resolve macromolecular size distributions at high concentrations, by simultaneously accounting for average mutual hydrodynamic and thermodynamic interactions. It offers high resolution and sensitivity of protein solutions up to 50?mg/ml, extending studies of macromolecular solution state closer to the concentration range of therapeutic formulations, serum, or intracellular conditions.

SUBMITTER: Chaturvedi SK

PROVIDER: S-EPMC6200768 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Measuring macromolecular size distributions and interactions at high concentrations by sedimentation velocity.

Chaturvedi Sumit K SK Ma Jia J Brown Patrick H PH Zhao Huaying H Schuck P P

Nature communications 20181024 1

In concentrated macromolecular solutions, weak physical interactions control the solution behavior including particle size distribution, aggregation, liquid-liquid phase separation, or crystallization. This is central to many fields ranging from colloid chemistry to cell biology and pharmaceutical protein engineering. Unfortunately, it is very difficult to determine macromolecular assembly states and polydispersity at high concentrations in solution, since all motion is coupled through long-rang ...[more]

PMID: 30356043

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Project description:Monoclonal antibodies represent the most important group of protein-based biopharmaceuticals. During formulation, manufacturing, or storage, antibodies may suffer post-translational modifications altering their physical and chemical properties. Such induced conformational changes may lead to the formation of aggregates, which can not only reduce their efficiency but also be immunogenic. Therefore, it is essential to monitor the amount of size variants to ensure consistency and quality of pharmaceutical antibodies. In many cases, antibodies are formulated at very high concentrations > 50 g/L, mostly along with high amounts of sugar-based excipients. As a consequence, all routine aggregation analysis methods, such as size-exclusion chromatography, cannot monitor the size distribution at those original conditions, but only after dilution and usually under completely different solvent conditions. In contrast, sedimentation velocity (SV) allows to analyze samples directly in the product formulation, both with limited sample-matrix interactions and minimal dilution. One prerequisite for the analysis of highly concentrated samples is the detection of steep concentration gradients with sufficient resolution: Commercially available ultracentrifuges are not able to resolve such steep interference profiles. With the development of our Advanced Interference Detection Array (AIDA), it has become possible to register interferograms of solutions as highly concentrated as 150 g/L. The other major difficulty encountered at high protein concentrations is the pronounced non-ideal sedimentation behavior resulting from repulsive intermolecular interactions, for which a comprehensive theoretical modelling has not yet been achieved. Here, we report the first SV analysis of highly concentrated antibodies up to 147 g/L employing the unique AIDA ultracentrifuge. By developing a consistent experimental design and data fit approach, we were able to provide a reliable estimation of the minimum content of soluble aggregates in the original formulations of two antibodies. Limitations of the procedure are discussed.

Dataset Information

Measuring macromolecular size distributions and interactions at high concentrations by sedimentation velocity.

Publications

Measuring macromolecular size distributions and interactions at high concentrations by sedimentation velocity.

Similar Datasets

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